Centrosome Linker-induced Tetraploid Segregation Errors Link...
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Centrosome Linker-induced Tetraploid Segregation Errors Link 1
Rhabdoid Phenotypes and Lethal Colorectal Cancers 2
3 Andrea Remo
1†, Erminia Manfrin
2†, Pietro Parcesepe
2†, Alberto
Ferrarini
3, Hye Seung Han
4, 4
Mickys Ugnius5, Carmelo Laudanna
6, Michele Simbolo
2, Donatella Malanga
6, Duarte Mendes 5
Oliveira6, Elisabetta Baritono
1, Tommaso Colangelo
7, Lina Sabatino
8, Jacopo Giuliani
1, Enrico 6
Molinari1, Marianna Garonzi
9, Luciano Xumerle
9, Massimo Delledonne
9,10, Guido Giordano
11,12, 7
Claudio Ghimenton2 Fortunato Lonardo
13, Fulvio D’angelo
14, Federica Grillo
15, Luca Mastracci
15, 8
Giuseppe Viglietto6, Michele Ceccarelli
8,14, Vittorio Colantuoni
8, Aldo Scarpa
2,16* and Massimo 9
Pancione8,17*
. 10 11 1Pathology Unit, “Mater Salutis” Hospital AULSS9, Legnago (Verona), Italy;
2 Department of 12
Diagnostics and Public Health – Section of Pathology, University and Hospital Trust of Verona, 13 Verona, Italy;
3 Menarini Silicon Biosystems S.p.A, Bologna, Italy,
4Department of Pathology, 14
Konkuk University School of Medicine, Seoul, Korea; 5National Center of Pathology, Affiliate of 15
Vilnius University Hospital Santariskiu Clinics, Vilnius, Lithuania; 6
Department of Experimental 16 and Clinical Medicine "Gaetano Salvatore", University “Magna Grecia”,Catanzaro, Italy;
7Institute 17
for Stem-cell Biology, Regenerative Medicine and Innovative Therapies (ISBReMIT), Casa 18 Sollievo della Sofferenza-IRCCS, San Giovanni Rotondo, Italy;
8Department of Sciences and 19
Technologies, University of Sannio, Benevento, Italy; 9Functional Genomics Center, Department of 20
Biotechnology, University of Verona, Verona, Italy; 10
Personal Genomics S.r.l., Verona, Italy; 21 11
CRO Aviano National Cancer Center, Aviano, Italy; 12
Medical Oncology Unit, San Filippo Neri 22 Hospital, Rome, Italy;
13Medical Cytogenetics and Molecular Genetics Unit, AORN "Gaetano 23
Rummo," Benevento; 14
Bioinformatics Laboratory, BIOGEM scrl, Ariano Irpino, Avellino, Italy; 15
24 Department of Surgical and Diagnostic Sciences (DISC), University of Genova and S. Martino 25 Polyclinic Hospital, Genova, Italy;
16ARC-Net Centre for applied research on cancer, University 26
and Hospital trust of Verona, Verona, Italy; 17
Department of Biochemistry and Molecular Biology 27 II, Faculty of Pharmacy, Complutense University, Madrid, Spain. 28 29 † These authors contributed equally to this work. 30
31 *Correspondence should be addressed to: Massimo Pancione, ([email protected]) 32
Department of Sciences and Technologies, University of Sannio, Via Port’Arsa, 1182100 33 Benevento, Italy; Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, 34 Complutense University, Madrid, Spain. Phone: +39 0824 305157; +34913941785 Fax +39 0824 35 305147 or Aldo Scarpa, ([email protected]), ARC-NET Research Centre, Policlinico GB 36 Rossi, Piazzale L.A. Scuro, 10, Phone.: +39 045 8124043, Fax:+39 045 8127432, 37 38 Running title: Centrosome Cohesion Anomalies and Cancer via CROCC Defects 39 40 Key words: Centrosome, rhabdoid Colorectal cancer, Aneuploidy, tumor heterogeneity 41 42 DISCLOSURE OF COMPETING INTERESTS: The authors declare that they have no 43 competing interests. 44 45 46 47 48
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Abstract 51
Centrosome anomalies contribute to tumorigenesis but it remains unclear how they are generated in 52
lethal cancer phenotypes. Here, it is demonstrated that human microsatellite instable (MSI) and 53
BRAF(V600E) mutant colorectal cancers with a lethal rhabdoid phenotype are characterized by 54
inactivation of centrosomal functions. A splice site mutation that causes an unbalanced dosage of 55
rootletin (CROCC), a centrosomal-linker component required for centrosome cohesion and 56
separation at the chromosome 1p36.13 locus, resulted in abnormally shaped centrosomes in 57
rhabdoid cells from human colon tissues. Notably, deleterious deletions at 1p36.13 were recurrent 58
in a subgroup of BRAF(V600E) mutant and microsatellite stable (MSS) rhabdoid colorectal cancers 59
but not in classical colorectal cancer or pediatric rhabdoid tumors. Interfering with CROCC 60
expression in near-diploid BRAF(V600E) mutant/MSI colon cancer cells disrupts bipolar mitotic 61
spindle architecture, promotes tetraploid segregation errors resulting in a highly aggressive 62
rhabdoid-like phenotype in vitro. Restoring near-wild-type levels of CROCC in a metastatic model 63
harboring 1p36.13 deletion results in correction of centrosome segregation errors and cell death, 64
revealing a mechanism of tolerance to mitotic errors and tetraploidization promoted by deleterious 65
1p36.13 loss. Accordingly, cancer cells lacking 1p36.13 display far greater sensitivity to 66
centrosome spindle pole stabilizing agents in vitro. These data shed light on a previously unknown 67
link between centrosome cohesion defects and lethal cancer phenotypes providing new insight into 68
pathways underlying genome instability. 69
Implications 70
Mis-segregation of chromosomes is a prominent feature of chromosome instability and intra-71
tumoral heterogeneity recurrent in metastatic tumors for which the molecular basis is unknown. The 72
present study provides insight into the mechanism by which defects in rootletin, a centrosome linker 73
component causes tetraploid segregation errors and phenotypic transition to a clinically devastating 74
form of malignant rhabdoid tumor. 75
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INTRODUCTION 77
The century-old hypothesis on the relationship between centrosomes and cancer, formulated by the 78
German embryologist Theodor Boveri more than 100 years ago (1,2), remains unanswered. 79
Centrosome abnormalities, consisting usually in increased numbers, are common in human tumours 80
(3), and experimentally induced tetraploid cells from extra centrosomes can be critical for 81
aneuploidy and metastatic progression of malignancy (3,4). However, insufficient progress has been 82
made in our knowledge on genetic defects underlying centrosome anomalies in tumourigenesis (1-83
4). In this scenario, the rare and lethal pathological variant of common colorectal cancers showing 84
rhabdoid phenotype (5-7), is of particular interest as it features recurrent mitotic anomalies of 85
enigmatic origin (8-10). We thus hypothesized that the systematic study of rare rhabdoid colorectal 86
cancers (RC), could provide insights into biological mechanisms responsible for the generation of 87
genome instability and reveal key factors for the development of aggressive disease entities. To test 88
this idea, we performed whole exome sequencing of two RC and discovered an enrichment of 89
centrosome anomalies and inactivation of ciliary rootlet coiled-coil (CROCC) gene (11,12), a 90
structural component of the centrosome linker which assembles and keeps connected the two 91
centrioles. Centrosomal alterations were assessed in an expanded series of rare RCs and related 92
tumours, and functionally characterized in colorectal cancer cellular models. 93
94 95 MATERIALS AND METHODS 96 97 Materials and Methods and any associated references as a continuation of the main text are 98
described more in detail within the supplementary material. 99
Patient and tissue cohort. 100
This study was conducted in accordance with Declaration of Helsinki ethical guidelines. It was approved by 101
an institutional review board, approval n. 997CESC from the Ethics Comittee (Comitato Etico di Verona e 102
Rovigo dell’Azienda Ospedaliera Universitaria Integrata) on 7 September 2016, documented by the CESC 103
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prot. 42160 on 9 September 2016 and formalized by the General Manager with deliberation n. 458 of 16 104
September 2016, communicated with protocol 51319 on 23 September 2016. 105
Formalin-fixed paraffin-embedded (FFPE) samples from 7 cases of primary rhabdoid colorectal 106
cancers (RC) and matched normal colonic mucosa were studied (cases RC1 to RC7 Supplemental 107
Table 1). Moreover, an independent validation series to screen the mutational status of newly 108
identified genes was analyzed (cases RC8 to RC12 Supplemental Table 1). FFPE samples from 7 109
rhabdoid tumours arised in central nervous system of patients between 2 months and 19 years of age 110
were collected from the files of the Azienda Ospedaliera Universitaria Integrata, Verona, Italy 111
These pediatric/young adults rhabdoid tumours are indicated as Rhabdoid of infancy (RI) 112
throughout the article. Two independent datasets of patients with classic type sporadic colorectal 113
cancer were analyzed: Dataset A including 141 primary cancers and Dataset B including 102 114
primary cancers. 115
Cell lines. 116
Human colon cancer cell lines HCT116, HT29, CaCo-2, LoVo, RKO, T84, DLD1, SW480 and 117
SW620 were purchased from American Type Culture Collection (ATCC). BJ human fibroblasts and 118
G401 cells derived from normal foreskin and pediatric rhabdoid tumour were used as a non-119
neoplastic control and a pure rhabdoid model, respectively. 120
Whole-Exome Sequencing. 121
Whole-exome sequencing with 100-bp paired reads was performed with a HiSEQ1000 (Illumina), 122
using 1.3 µg genomic DNA (based on fluorometric Picogreen dsDNA quantification) and 123
enrichment for whole exome according to TruSeq Exome Enrichment Guide (Illumina). 124
Functional in vitro assays 125
RKO cells were transiently transfected with SureSilencing control or CROCC shRNA expression 126
plasmids KH23140P (Qiagen) containing the puromycin resistance cassette. After selection 127
puromycin (Thermofisher), single colonies were amplified and assessed for efficient CROCC 128
silencing by quantitative PCR (qPCR) and western blot, respectively. HT29 and T84 cells were 129
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transfected with the full-length CROCC coding sequence or a truncate form (1–494aa) cloned with 130
GFP epitope or GFP alone (used as control). For long-term experiments CROCC-GFP+ cells were 131
maintained in 0.6 mg/ml of G418. 132
Statistical analysis 133
Data are presented with mean, medians and ranges. The P values were calculated two sided. 134
Statistical analyses were conducted by GeneSpring R/bioconductor v.12.5 and R based package, 135
SPSS v15 and GraphPad Prism 5. 136
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RESULTS 139
Discovery of CROCC mutations and centrosome anomalies 140
Two previously reported primary BRAFV600E
mutant RCs (RC1 and RC2) (9,10), harboring 141
microsatellite instability due to defective DNA mismatch repair (MMR) machinery caused by 142
promoter methylation of the MLH1 gene, were subjected to whole exome sequencing (WES) using 143
DNA from formalin-fixed paraffin-embedded (FFPE) matched tumour/normal samples 144
(Supplemental Table 1). We detected an exceptionally large number of somatic point mutations, 145
1056 and 1078 per 106
bases for RC1 and RC2, respectively, which is consistent with the presence 146
of MMR defects (13-15) (Figure 1a). About 1/5 of mutations occurred within CpG dinucleotide 147
context as seen in classical colorectal cancers (14). Transitions were more frequent than 148
transversions (71.8% vs 28.2%, Supplemental Fig. 1a) with a dominance of C>T/G>A, T>C/A>G 149
transitions (Supplemental Fig. 1b) which is characteristic of the mutational signature due to 150
alterations of MMR mechanisms (signature 6) (13). The most prevalent single nucleotide variants 151
(SNVs) were non-silent mutations (14), where over 90% of potentially damaging mutations were 152
missense and around 10% were splicing, stop-gain, stop-loss or, rarely, frameshift insertions or 153
initiation codon mutations (Figure 1a and Supplemental Fig. 1c,d). The two RC cases shared 112 154
(10%) mutated genes (Supplemental Fig. 2a). By applying DrGaP computational tool (16), which 155
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allows to infer cancer driver genes, a number of potential candidate disease-causing genes were 156
identified (Supplemental Table 2), ~half of which (45%), were enriched for 157
cytoskeleton/centrosome and microtubule biological functions (Supplemental Fig. 2b,c). 158
The search of candidate genes in the cancer genome atlas (TCGA) database (13-15) 159
comprising 224 sequenced classical colorectal cancers (http://www.cbioportal.org) revealed that the 160
majority (65%) of the candidates had a low frequency of mutations (≤4% of cases). Strikingly, 161
among these, only for CROCC gene mapping to 1p36.13 (11,12) involved in centrosome cohesion 162
and disjunction, no somatic mutations (0/224; 0%) were reported (Figure 1b). Notably, 1p36 163
deletions are recurrent in neuroblastoma, Wilms' tumor and medulloblastoma. In our two RC cases, 164
CROCC harbored two missense mutations, p.Ala161Ser (c.481G>T, Exon 4) and p.Val1885Ala 165
(c.5654T>C, Exon 35), and one prominent splicing mutation at the conserved 3’ acceptor splice site 166
(c.3705-2A>G) in the intron between exons 25-26 (Figure 1c). A review of multiple CRC 167
sequencing datasets (N=2070) revealed CROCC mutations in (1,4% of cases). However, although 168
of unknown significance none of the CROCC mutations was identified as putative driver mutations 169
in colorectal cancer (Supplemental Fig. 2d). SMARCB1 and SMARCA4 mutations, which have 170
been associated with rhabdoid phenotype (6), showed a trend of mutual exclusivity with CROCC 171
alterations. However, only putative truncating driver mutations in SMARCB1 and/or SMARCA4 172
correlated with tumour poor differentiation and short-time metastatic progression. Therefore, we 173
reasoned that the splicing mutation detected in RC1 might be causally correlated with rhabdoid 174
phenotype. Indeed, the mutation reduced the strength of the physiologic acceptor site, causing a 175
large deletion of the CROCC coding region involving exons 23-31 (17,18) (Supplemental Fig. 3a). 176
Independent RT-PCR-derived products spanning exons 5-7 and 33-35 showed that the expression 177
of CROCC in the tumour samples RC1 containing the splicing mutation was reduced, thus 178
suggesting the alteration of the mature transcript by the utilization of cryptic splice sites or by the 179
activation of the nonsense-mediated mRNA decay pathway, which impair transcripts harboring 180
large deletions (Supplemental Fig. 3b) (17). Unexpectedly also RC2, displayed expression levels 181
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in tumour lower than in normal tissue, supporting a role for defective CROCC expression in 182
rhabdoid tumours (Figure 1d). We next used immunohistochemistry and immunofluorescence at 183
high magnification with an anti-CROCC antibody to analyze the centrosomes in rhabdoid cancers 184
and matched normal tissues (Supplemental Table 3). We found that nearly 50% of tumour cells 185
had no CROCC immunolabelling, and the presence of cells with a single and often abnormally 186
shaped, larger (up to 6-fold greater than normal) or fragmented centrosomes, suggesting the 187
presence of numerical and structural centrosome aberrations (Supplemental Fig. 3b). We also 188
observed dramatic and uncommon cytological defects, such as anucleated cells having larger 189
centrosomes positive for CROCC associated to mitotic catastrophe in late telophase particularly in 190
RC1 harboring the splicing site mutation (Figure 1d). We used the pericentriolar material (PCM) 191
component γ-tubulin, as our reference marker for immunolabelling experiments, because it 192
consistently colocalizes with centriole markers which are closely connected in interphase by the 193
centrosomal linker (1,2,11,12). Moreover, γ-tubulin has been proposed as a marker to identify 194
spindle poles (19,20). We observed a remarkable loss of cell polarity in interphase nuclei and 195
abnormal mitotic figures many of which included asymmetric bipolar or monopolar spindles. 196
Rhabdoid cells showed a diffuse staining of γ-tubulin into the cytoplasm and reduced centrosomal 197
localization, a phenomenon described in tumors with high metastatic potential (19) (Supplemental 198
Fig. 3c,d). Double immunofluorescence analysis using antibodies directed against CROCC and γ-199
tubulin confirmed these observations and revealed cells either with fragmented/larger centrosomes 200
or with a consistent loss of centrosome staining (Supplemental Fig. 3). These experiments 201
indicated that genetic defects in CROCC and other centrosome components may compromise 202
centrosome function in RC. 203
A validation set of 10 additional rhabdoid colorectal cancers was studied, including 3 cases 204
(RC7, RC9, RC11) with microsatellite instability due to MLH1 promoter methylation and 7 cases 205
(RC3-6, RC8, RC10, RC12) with stable microsatellites (Figure 2a and Supplemental Table 1). 206
Targeted sequencing identified three CROCC mutations (p.Ser1320Ile, p.Arg1659His and 207
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p.Ala1510Thr) of unknown significance in additional 2 cases (RC9 and RC11) harboring 208
microsatellite instability (1,2,11,12). Indeed, the 24 CROCC mutations identified across cbioportal 209
database were more recurrent in MSI (12/24; 50%) than in MSS (6/24; 25%) CRCs. Notably, 210
CROCC mutations were classified as missense (n=22) or truncating mutations (n=2) of unknown 211
significance but associated with both well- differentiated and early-stage classical CRCs. In our RC 212
dataset, 5 of the remaining cases for which sufficient material was available (cases RC3 to RC7) 213
harbored loss of heterozygosity (LOH) at the 1p36.13 locus, where CROCC resides, which was 214
associated to mRNA below normal levels (Figure 2b). In keeping with the findings in RC1 and 215
RC2 cases, comparable levels of centrosomes defects and a high prevalence of bizarre mitotic 216
figures and/or cytomorphologic aberrations were evident in all tumours (Figure 2c and 217
Supplemental Table 3). Analysis of independent CRC databases (N=1387) for which copy number 218
alterations were available (http://www.cbioportal.org) revealed no alteration at 1p36.13 locus, 219
suggesting CROCC impairment as a consequence of reduced gene dosage (21) caused by allelic 220
deletion. As centrosome anomalies are intimately connected with chromosome segregation errors 221
(1,3,20), we assessed DNA content in tumour samples (cases RC1 to RC7, Supplemental Table 222
3). Remarkably, we observed recurrent ploidy abnormalities mainly consisting of triploid or near-223
tetraploid cells ranging from 10% to 40% of tumour cells (Figure 2d). Globally, these results 224
indicated that centrosome defects underlie RC pathogenesis. 225
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Centrosome and genomic profiling of rhabdoid tumours of infants 227
Insight into genetic characterization of rhabdoid neoplasms are limited to the so called 228
extrarenal rhabdoid tumours arising in children, in which inactivating mutation and/or deletion of 229
the chromatin remodelling gene SMARCB1 (INI1) and low mutation load have been reported (6,22-230
24). We analyzed 7 cases of this tumour type, hereafter named Rhabdoid of infant (RI), for 231
centrosome and molecular anomalies (Figure 3a and Supplemental Table 4). Compared to RCs, 232
analysis in pediatric tumours was associated with much higher CROCC mRNA expression levels 233
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than matched normal tissues (P<0.00001; Figure 3b). Target-sequencing identified no mutations (0 234
of 7 tumours) in the CROCC gene. Moreover, CROCC immunostaining was seen as a single and 235
large signal adjacent to the nuclei in almost the totality of the cells (80%), only 10% of cells had no 236
centrosome staining (Figure 3b and Supplemental Table 4). Consistent with literature, the 237
genetic profile of RI revealed missense or truncating mutations in SMARCB1 (5/7, 71%) (6,23,24) 238
and/or TP53 (3/7, 42%) accompanied by a near diploid DNA content and less aggressive clinical 239
course when compared to RCs (Figure 3c-e). This suggested that RIs, which are characterized by 240
SMARCB1 (INI1) mutation, did not harbor any CROCC alteration and did not display the 241
centrosomal defects observed in RC. Inspection of an available database from pediatric rhabdoid 242
cells (25) confirmed that both mutations or genetic deletion affecting CROCC locus were infrequent 243
(2/20, 10%), whereas the transcript profile tended to be similar to our RI dataset (Figure 3c). 244
Therefore, rhabdoids arising in colorectal cancer although morphologically indistinguishable from 245
their pediatric counterparts demonstrate distinct molecular, cytogenetic and centrosomal 246
aberrations. 247
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Centrosome and CROCC expression in classical colorectal cancers 249
We screened 242 primary classic colorectal cancers (CRCs), comprising two independent 250
series of 140 (dataset A) (26,27) and 102 (dataset B) (28,29) cases for CROCC mRNA and protein 251
expression (Supplemental Table 5). CROCC mRNA expression levels were higher in CRCs than in 252
normal colonic mucosa. However, no significant protein expression change in tumour tissues compared to 253
that in normal mucosae was detected (Supplemental Fig. 4a). In cohort A, using 254
immunohistochemistry and immunofluorescence against CROCC and γ-tubulin, we found round 255
and uniform in size centrosomes, prevalently in normal number (1-2 per cell; 97/140, 69.3%), 256
supernumerary (>2 per cell; 39/140, 27.8%) and only few (<1 per cell; 4/140, 2.9%) displayed 257
reduced centrosome labeling (Supplemental Fig. 4b). Centrosome abnormalities, particularly 258
supernumerary centrosomes were more prevalent in advanced stage (stage III-IV) than in low stage 259
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(stage I-II) lesions (Supplemental Fig. 4c). In cohort B which was enriched for stage III-IV 260
tumours (79.5% of cases), we confirmed a high prevalence of supernumerary (60/102; 59%) 261
(1,3,20) or defective (11/102; 11%) centrosomes associated to poorer clinical course than those 262
expressing a normal pattern (31/102; 30%, HR=0.30; 95% CI (0.21-0.81); P<0.0001) in keeping 263
with the notion that numerical centrosomal abnormalities are more common in invasive cancers 264
(1,3,20) (Supplemental Fig. 4c). To independently validate the pattern of gene expression changes 265
detected in our datasets, we analyzed the patient-matched tumour-normal expression data available 266
from the TCGA (14) and three independent datasets GSE20916 (30), GSE41258 (31), GSE30540 267
(32) of classical colorectal cancer (Supplemental Fig. 4d). CROCC mRNA was up-regulated in 268
CRC compared to normal only in TCGA database. The analysis of other datasets revealed an 269
heterogeneous expression pattern, while CROCC up-regulation compared to normal control not 270
reached statistical significance. The analysis of GSE30540 (32) dataset, for which both 271
transcriptomic data and degree of chromosome instability (CIN) were available, revealed that 272
CROCC expression levels tended to be lower in CIN-high than in CIN-low tumours (Supplemental 273
Fig. 4d). These data suggested thus the possibility that imbalanced genetic defects at CROCC locus 274
may be related to marked anomalies in the fidelity of chromosome segregation. 275
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CROCC depletion impairs mitosis and induces rhabdoid-like features 277
We next sought a genetic basis for the relation between CIN and rhabdoid phenotype by 278
examining whole exome sequencing and transcriptomic data from The Cancer Cell Line 279
Encyclopedia (CCLE) (25). Unexpectedly, data from a collection of 60 CRC cell lines revealed that 280
the deletions at 1p36.13 locus tended to be more prevalent in CIN-high (23.6%, 9 of 38) compared 281
to CIN-low (9.1%, 2 out of 22) cells (Supplemental Fig. 5a). However, cell lines with 1p36.13 282
deletion displayed neither rhabdoid phenotype nor BRAF mutations. As expected, compared to 283
cells retaining 1p36.13 locus, those harboring the deletion revealed a gene-expression signature 284
significantly enriched for pathways implicated in chromosomal instability (33,34) (Supplemental 285
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Fig. 5b,c). In a panel of CRC cells, we then confirmed that both CROCC mRNA and protein 286
expression levels were concordant and higher in CIN-low than in CIN-high cell lines (P<0.05, 287
Supplemental Fig. 6a). CIN-low cells showed centrosomes stained for CROCC and γ-tubulin that 288
were structurally indistinguishable from those in normal human fibroblasts BJ, consistent with 289
literature (35) (Supplemental Fig. 6a,b). In line with this, CIN-high displayed a higher frequency 290
of micronuclei and nuclear γH2AX foci than CIN-low cells (29) (Supplemental Fig. 6c,d). This 291
suggested that CROCC might be a CIN-suppressor gene and its deletion in CIN-low/BRAF mutant 292
cell lines might be permissive for abnormal phenotypes. Therefore, we reasoned that RKO cells, 293
sharing a near-diploid karyotype, BRAF(V600E)
mutation/MSI and alterations in 294
microtubule/centrosomal components with RC (14), could be an excellent system to explore 295
CROCC silencing in vitro. We found that the clone sh4, hereafter named CROCCKD
, provided a 296
stable and consistent knockdown of CROCC transcript to more than 75% and protein to 3.3-fold 297
lower then RKO cells transfected with control vector (shCon), achieving nearly comparable levels 298
to those seen in vivo (Supplemental Fig. 7a). Previous studies have demonstrated that CROCC 299
knockdown in non-transformed cells causes centriole splitting and increases centrosome separation 300
(11,12). By using γ-tubulin and centrin as reference markers, we observed a consistent PCM 301
fragmentation after CROCC depletion which resulted in abnormal chromosome segregation and 302
higher frequency of monopolar spindles as compared to control (Figure 4a and Supplemental Fig. 303
7b). Importantly, monopolar spindles displayed larger or “fragmented” centrosomes which 304
accounted for 85% of the abnormal phenotype (Figure 4a-c and Supplemental Fig. 7c). Thus, 305
BRAF-mutant/MSI CRC cell lines in which centrosome and microtubule stability is damaged by 306
genetic hypermutation, CROCC depletion may determine a major impact in the progression of 307
mitotic errors (36-38). Consistently, an increased frequency of micronuclei (median 11% 308
CROCCKD
versus 1% ShCon cells P=0.0003) and γH2AX nuclear foci (median 43% CROCCKD
309
versus 18% ShCon cells P=0.011 were observed (Figure 4c, and Supplemental Fig. 7d). 310
Metaphase karyotyping revealed that CROCC deficiency leads to an increased number of tetraploid 311
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(4N) cells (median 13.3% CROCCKD
versus 3.51% ShCon cells P=0.001) characterized by 312
prominent and larger nuclei than diploid (2N) cells. Consistently, analysis of centromeric probes in 313
intephase nuclei confirmed tetraploidy (Figure 4c). The number of CROCC-deficient cells was 314
reduced in G0/G1 or G2/M phases when compared to the wild-type population (by 26–45%, FACS 315
analysis), suggesting an impaired cell cycle progression as a consequence of misaligned 316
chromosomes (1-3,33) (Supplemental Fig. 7d). By contrast, cells grown under replication stress 317
conditions (serum deprivation) resulted in higher proliferation rate than control cells (34) (Figure 318
4d). Most strikingly, CROCC-deficient cells exhibited all cardinal signs of rhabdoid features (8-10), 319
displaying huge nuclei pushed to the periphery of the cells with single or multiple large nucleoli 320
associated with eosinophilic cytoplasmic inclusions and large cellular protrusions resembling the 321
morphology observed in vivo (Figure 4d and Supplemental Fig. 8a). These features resulted in 322
the activation of pro-metastatic genes involved in epithelial mesenchymal transition accompanied 323
by a dramatic change of spindle-shaped morphology (4,7) consistent with the enhanced metastatic 324
potential of rhabdoid phenotype (Supplemental Fig. 8a,b). Expression of exogenous green 325
fluorescent protein (GFP)-tagged CROCC (1–2018aa) rescued these phenotypic changes induced by 326
depletion of endogenous CROCC (Supplemental Fig. 8b) (11). In line with previous results 327
(11,12), we observed no alteration in cell cycle profile or aberrant phenotypic changes after CROCC 328
depletion in non-transformed BJ cells. Therefore, CROCC depletion in BRAF-mutant near-diploid 329
cancer cells induces tetraploidization and rhabdoid phenotype in vitro. 330
331
Tolerance to mitotic errors and tetraploidization promoted by 1p36.13 deletion 332
As colorectal cancer cells with driver mutations in CROCC have not been reported, to test the 333
hypothesis that CROCC impacts tumour growth and centrosome-related mitotic errors, we analyzed 334
metastatic colorectal cancer T84 cells harboring an allelic deletion at 1p36.13 locus (25). Although 335
T84 are well-differentiated cancer cells and do not show rhabdoid morphology, they however 336
exhibit some of the characteristics detected in RKO CROCC-depleted cells. Consistent with 337
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13
reduced CROCC endogenous activity, we observed an increased rate of micronuclei, tetraploid or 338
near-tetraploid cells and recurrent mitotic errors resulting essentially in “monopolar spindles, which 339
were more recurrent under replication stress conditions (Figure 5a and Supplemental Fig. 8c,d). 340
We then investigated the localization of CROCC in the centrosome by immunofluorescence. 341
Almost half of the cells (40%) revealed a faint CROCC signal, which was consistently accompanied 342
by atypical γ-tubulin aggregates prevalently in cells with mitotic anomalies (Figure 5a). Most 343
strikingly, such aberrations were rarely, if ever, detected in pediatric rhabdoid G401 or colon cancer 344
cell lines with an intact 1p36.13 locus (Supplemental Fig. 8c,d). Therefore, we transfected 345
CROCC-GFP and GFP alone (control) into T84 cells. Restoration of CROCC, determined a 346
dramatic decrease of cell viability (12 days later, 0%) as compared to control plasmid. Similarly, 347
we detected a higher number of G0/G1 cells than control (41% vs 26%; P=0.0018, Figure 5b,c). 348
Gain of CROCC conferred a flat/adherent phenotype and formation of filament-like structures co-349
localizing with γ-tubulin resulting in an expression of mesenchymal genes lower than in control (4) 350
(Supplemental Fig. 9a). Accordingly, we detected a 4-fold decrease of tetraploid cells, and reduced 351
γH2AX foci from 59% to 22% with respect to control cells (Figure 5c) raising the possibility that 352
the centrosome spindle pole integrity is strongly affected by 1p36.13 deletion. To see if T84 cells 353
are sensitive to mitotic drugs, we mined the data from the Genomics of Drug Sensitivity in Cancer 354
project (Sanger panel). As shown in (Supplemental Fig. 9b), among 221 molecules tested, IGF1-R 355
inhibitor (linsitinib) and Epothilone B a microtubule stabilizing agent, were significantly effective 356
in T84 lines. Accordingly, we observed a significant difference in the sensitivity to Epothilone B in 357
1p36.13 deleted cells as compared to cells with an intact 1p36.13 locus. Similar results were not 358
reproduced comparing CIN-low and CIN-high CRC cell lines (Supplemental Fig. 9b). We used 359
another cell line HT29 with an intact 1p36.13 locus to test CROCC restoration. Similarly to T84, 360
we observed a significant decrease of micronuclei in HT29-CROCC-GFP+ cells compared to 361
control. Although gain of CROCC in HT29 increased the cell death, it appeared an essential gene 362
only for T84 cell survival lacking 1p36.13 (Supplemental Fig. 9c). This supported the hypothesis 363
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14
that a reduced CROCC dosage promotes defects in spindle-assembly checkpoint. When we repeated 364
the experiments using a GFP-tagged truncated form of CROCC (1–494aa) (11), we observed that 365
this mutant failed to rescue the aberrant growth phenotype and mitotic errors (Supplemental Fig. 366
9d). Consistent with previous findings (11), we did not detect phenotypic changes in non-367
transformed BJ cells transduced with the full-length CROCC construct. Thus, we conclude that 368
DNA segregation errors resulting from impaired centrosome function are driven by reduced 369
CROCC dosage at 1p36.13 locus. 370
371
DISCUSSION 372
Our understanding of the molecular architecture and function of centrosomal linker 373
components in physiological and pathological processes remain rudimentary. Besides CROCC 374
(Rootletin), multiple proteins including C-NAP1 (CEP250), CEP68 and LRRC45 have been 375
implicated in centrosome linker formation and function. CROCC is able to maintain centrosome 376
cohesion in part through inhibition of VHL-mediated Cep68 degradation (36). It has recently been 377
proposed that a vast network of repeating CROCC units with C-Nap1 as ring organizer and CEP68 378
as filament modulator forms the centrosome linker structure (37). We show here that genetic 379
deletion in CROCC, leads to centrosome anomalies resulting in tetraploid DNA segregation errors, 380
providing insights into mechanism by which genome instability contributes to lethal cancers for 381
which no therapies are available (Figure 5d). In addition, we show that rhabdoid colorectal cancers 382
are not genetically related to their pediatric counterparts (22-24), in which we find recurrent 383
SMARCB1 gene alterations but no evidence of centrosome anomalies. Previous studies have 384
revealed that driver genes implicated in human cancer (3,4) can promote centrosome over-385
duplication (2,20) which through whole genome doubling facilitates chromosomal instability, 386
especially in metastatic tumours (4,38). However, an important drawback of these studies (3,4) is 387
that the mechanism of tetraploidization and underlying biological causes have remained unresolved. 388
We provide evidence that centrosome linker genes might be altered due to imbalanced genetic 389
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15
defects, interfering with protein complexes required for the correct assembling of spindle functions 390
in colorectal cancer cells (39). 391
Recently, factors involved in the stabilization and nucleation of microtubules around 392
kinetochores have been described in BRAF mutant colorectal cancer cells, highlighting the potential 393
to make these tumours vulnerable to microtubule-destabilizing anticancer drugs (40). Other studies 394
have showed that the centrosomal linker genes and microtubule motor proteins cooperate to keep 395
unlinked centrosomes in relative close proximity (41). Therefore, cumulative defects in these 396
pathways may result in spindle perturbations, providing an explanation for the observed mitotic 397
errors after CROCC depletion. The frequency of CROCC mutations in other tumors with MSI is 398
unknown. However, exploration of cbioportal database revealed a prevalence of CROCC mutations 399
in cancers with high mutational load. In contrast, 1p36.13 deletions appeared to be characteristic of 400
liver, skin or uterine carcinosarcoma with high levels of genomic instability. 401
Our findings underline that in CIN negative cancer cells with functionally compromised 402
centrosomes (i.e BRAF mutant CRC cells), CROCC depletion leads to monopolar spindle DNA 403
segregation defects exacerbating mitotic errors and promoting rhabdoid morphology. Therefore, 404
upregulation of CROCC in classical CRC particularly in MSI tumours, may provide a mechanism 405
of protection to potentially deleterious genetic changes (39,40). CROCC restoration in a metastatic 406
model with 1p36.13 deletion confirmed its role as a biological barrier against mitotic errors. In 407
agreement with this, colon cancer cells with 1p36.13 deletion display have increased sensitivity in 408
vitro to microtubule stabilizing agents used in pediatric tumors (42). However, we were unable to 409
demonstrate the detailed molecular mechanism by which independent CROCC defects promote 410
gross mitotic errors. In addition, other factors not present in our current models can influent 411
rhabdoid pathogenesis specially in MSS tumours. In fact, CROCC deletion was recurrent in CIN-412
High cancer cells without rhabdoid characteristic, supporting the concept that rhabdoid traits are 413
highly heterogeneous as consequence of multiple dysregulated developmental pathways. The RC 414
patients described in our study, presented lethal clinical outcomes with an average postoperative 415
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16
survival of only 7 months. Therefore, the recurrent CROCC genetic deletions identified in these 416
patients may be associated with the poor prognosis. From this point of view, identifications of new 417
molecular subgroups cannot be excluded. For example, we don’t know whether CROCC deletion is 418
a unfavorable prognostic only in BRAF mutant tumours or other subtypes with SMARC gene 419
mutations (SMARCB1, SMARCA4). So far, mutations in centrosome genes like CEP57, CEP135 420
and PLK4 kinase, have been only described in rare genetic disorders with genomic instability such 421
as microcephaly and Seckel syndrome (43,44). 422
Overall, our data uncover a mechanism by which defects of critical centrosomal components 423
cause unequal DNA segregation that contributes to the ongoing genetic heterogeneity in rare and 424
aggressive colon cancers. Our findings link for the first time centrosomal cohesion defects and 425
genomic instability, prompting for studies addressing how genetic centrosome anomalies are 426
connected with key pathways involved in safeguarding the integrity of the human genome. 427
428
429
430
431
432
ACKNOWLEDGMENTS: We thank, L. Cerulo, Department of Sciences and Technologies, 433
University of Sannio, Benevento, Italy; and G. Falco, Department of Biology, University of Naples, 434
Federico II, Naples, Italy for commenting on the molecular/clinical aspects of the manuscript and 435
for helpful discussions; Roberta Maestro (CRO, Aviano, Italy) for her kind gift of the BJ human 436
skin fibroblasts and G401 cells and for helpful discussions; Erich Nigg for his kind gift and 437
suggestions about clone 6150861 pEGFP Rootletin; ARC-NET Research Centre core imaging 438
facility for assistance with microscopy. T.C. is supported by a fellowship from Associazione 439
Italiana Ricerca sul Cancro (AIRC) (project code: 19548). This work was supported by Department 440
Funds of Mater Salutis Hospital, FUR and the Italian Ministry of University and Research (MiUR) 441
to M.P., and AIRC 5x1000 (n. 12182) to AS. 442
443
444
445
446
447
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448
449
450
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565 566 567 568 569 570 571 572 573 FIGURE LEGENDS 574 575 576 Fig. 1 Whole exome sequencing reveals mutations in CROCC, encoding an essential component of the centrosome 577 linker. 578 a Representative rhabdoid colorectal cancers histopathological images from RC1 and RC2 that were subjected to whole exome 579 sequencing (H&E, haematoxylin and eosin). The graph indicates the total number of somatic mutations per tumor. The circo shows 580 the distribution of non-silent mutations and copy number variations (CNVs) as indicated by the diverse colours; the outer ring 581 indicate the chromosomes. b Prevalence of alterations in the candidate genes harboring somatic mutations in both RC1 and RC2 in 582 224 colorectal cancers of the TCGA database. c CROCC chromosome localization (1p36.13) and organization (from Ensembl, 583 reference transcript ENST00000375541). All 37 exons, are depicted as green vertical bars and introns as horizontal lines. Solid 584 circles indicate the mutations identified in RC1 and RC2. The “proteinaceous linker” is composed of CROCC filaments (black 585 arrow) that physically connect the mother (M) and daughter (D) centrioles surrounded by the pericentriolar material (PCM). At the 586 onset of mitosis (Mi) the linker is disassembled to support the formation of the bipolar mitotic spindle. d Quantification of CROCC 587 mRNA (qPCR) expression levels in tumour and adjacent normal mucosa. Data are mean ± standard deviation (s.d); n=5 biological 588 replicates; **P<0.01, two-tailed Student’s t-test). Representative images of CROCC immunostaining in non-neoplastic colon mucosa 589 and a fallopian tube used as control (red arrow). CROCC immunopositive centrosomes are reduced in number (black arrow) or 590 mispositioned (distant/separated from the nucleus) in RC1, (inset modeled image). Scale bars are reported in each microphotograph. 591
592
593
594
595 Fig. 2 Centrosome anomalies characterize colorectal cancer with rhabdoid phenotype. 596 a Histopathological images from a subset of 5 additional prototypical rhabdoid colorectal cancers (RC), in which are evident rounded 597 eosinophilic cytoplasmic inclusions, eccentric nuclei and prominent nucleoli. Scale bar, 20 µm H&E images. b Mutations for 598 selected driver genes and CpG island methylation (CIMP) profile accompanied by loss of heterozygosity analysis at 1p36.13 locus. 599 CROCC mRNA (qPCR) expression levels in tumours and adjacent normal mucosa, Data are mean ± standard error of the mean 600 (s.e.m); (n=5 biological replicates, P*<0.05, **P<0.01, two-tailed Student’s t-test). c Representative immunohistochemical analysis 601 from case RC5: Cytokeratin-18 (CK18) marks intermediate filaments in an anucleated cell (red arrow); Ki67 reveals abnormal 602 chromosome structures (black arrow); CROCC marks a multinucleated cell (black arrow), anucleated cell (red arrow) or it appears 603 fragmented in a mitotic cell (monopolar spindle, green arrow). Scale bar, 10 μm. Right, quantification of the centrosome phenotypes 604 against CROCC observed in all RCs (n=2 experiments, >500 cells/sample). d Cytogenetic abnormalities (tetraploid signals, red 605 arrows) observed by fluorescence in situ hybridization (FISH) using the centromeric chromosome probes illustrated. Scale bars, 20 606 and 40 µm. Left, ploidy pattern in all RCs (for chromosomes 1, 12 and 17, n=2 experiments, >500 cells per sample). Right, 607 quantification of cells with polyploidy measured as ratio of triploid and tetraploid on diploid cells for each tumour. 608 609 610 611 612 613 Figure 3. Centrosome and cytogenetic aberrations comparison between colorectal and pediatric rhabdoid tumors. a 614 Representative haematoxylin and eosin (H&E) images of pediatric rhabdoid tumors. Scale bar, 50 µm. b Right, 615 immunohistochemical and interphase FISH analysis for the indicated markers. Note that centrosomes are single, larger, uniform in 616 size and close to the nuclei (black arrowhead). Left, quantification of CROCC immunohistochemistry (IHC) and centromeric (CEN) 617 signals (Chr 1 and Chr 17) in pediatric rhabdoid tumors, (>500 cells per tumor) were evaluated, percentages represent mean values 618 from three independent investigators. Quantification of CROCC mRNA (qPCR) expression levels. Each circle represents the mean 619 value of five biological replicates from a single lesion, **P<0.01, two-tailed Student’s t-test. c CROCC expression in pediatric 620 rhabdoid derived cancer cell lines according to copy number alterations and mutational load (Novartis/broad cancer cell lines 621 encyclopedia). d The panel shows the distribution of non-silent missense or truncating mutations for the indicated pathways in 622
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rhabdoid colorectal cancer (RC) and rhabdoid of infants (RI). e Kaplan-Meier overall survival curve for RI (age class 2 months–19 623 years) and RC (age class 49-83 years). The p value is obtained by the log-rank test is reported in the graph. 624 625 626 627 628 629 630 631 632 633
634 Fig. 4 CROCC depletion induces rhabdoid phenotype exacerbating DNA segregation errors. a Images of RKO cells 635 with stable CROCC depletion (CROCC KD) showing that in mitosis there is an abnormal spindle formation - “monopolar spindles” - 636 as compared to control (CON, left panel). Large (white arrow) or fragmented (green arrow) centrosomes are shown. Cells are stained 637 using immunofluorescent antibodies with different colors as indicated (αTUB, anti-alpha-tubulin antibody; γTUB, anti-beta-tubulin 638 antibody) and nuclei are stained with DAPI (49,6-diamidino-2-phenylindole). Scale bar, 5 µm. Below is a schematic illustration of 639 cells with aberrant spindles (85%). b anaphase bridges (white arrow), multinucleated (green arrow), multilobulated nucleus (red 640 arrow) and fragmented centrosomes (blue arrows) associated with loss of cell polarity and large micronuclei (white arrow) in 641 CROCC depleted cells. Scale bar, 5 µm. c The upper left graph shows the percentage of micronuclei and monopolar spindles (>250 642 cells per cell line, triplicate experiments, P**<0.01,***P<0.001 Mann–Whitney U test). Representative images of metaphase 643 chromosome spreads and cells stained with DAPI and anti-centromere antibody (ACA). Scale bar, 10 µm. The lower left graph 644 shows the quantification of ploidy content at metaphase. Data are mean ± standard error of the mean (s.e.m); (n=5 biological 645 replicates**P<0.01, two-tailed Student’s t-test). The lower right graph shows tetraploid on diploid cells ratio. d The upper graphs 646 report the survival assay with serum supplementation or under replication stress condition “serum deprivation”. Error bars represent 647 mean ± s.e.m, of five independent experiments *P<0.05, **P<0.01, ***P<0.001, two-tailed Student’s t-test. Below are 648 representative cytomorphological changes showing large polygonal cells and eccentric round nuclei with prominent nucleoli (black 649 arrow) and eosinophilic hyaline cytoplasmic inclusions (red arrow). Haematoxylin and eosin (H&E) pictures. 650 651 652 653 654 655 656 657 658 Figure 5. CROCC abrogates centrosome-related mitotic errors in 1p36.13 deleted cancer cells. a Images of T84 cells 659 with a large micronucleus (white arrow) and significantly reduced CROCC staining (enlarged in insets), anaphase bridges (green 660 arrow) or monopolar spindle (red arrow) associated to deficient or fragmented γ-tubulin dots (enlarged in insets). Scale bar, 5 μM. 661 The upper right graph reports the percentage of anaphase showing segregation errors and micronuclei in T84 cells with serum 662 supplementation or serum deprivation (SD) at 12h. Error bars represent mean ± standard error of the mean (s.e.m)*P<0.05, two-663 tailed Student’s t-test). b Representative images of T84 cells transfected with full-length human CROCC-GFP or GFP alone, 664 immunostained for γ-tubulin (red, enlarged in insets). The graph on the right shows the survival of T84 transfected with CROCC and 665 matched control cells maintained in neomycin selection (0.6 µg ml-1) for the indicated time. Viability was assessed by a colony 666 formation assay. The GFP vector was used as a control. Cells were fixed, stained, and photographed after 6 and 12 days of culture. c 667 The left graph reports flow cytometry analysis after 6 days. Error bars represent mean ± s.e.m, of five independent experiments, 668 **P<0.01, ***P<0.001, two-tailed Student’s t-test). Tetraploid on diploid cells ratio after 6 days quantified by metaphases spreads 669 (16 independent experiments for each condition, **P<0.01, two-tailed Student’s t-test). The right graph shows the percentage of 670 𝑦H2AX foci in prometaphase (>250 cells per cell line, ***P<0.001, Mann–Whitney U test). d Schematic representation of rhabdoid 671 colorectal cancer (CRC) progression. In CRC with defective microtubule functions, BRAF (V600E) mutation depletion of CROCC 672 causes defective centrosome structure, abnormal mitotic progression and lethal cancer phenotypes. 673 674 675 676 677 678 679
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Somatic mutations
Gain
Nonsynomous SNV
Splicing
Stopgain
Stoploss
Frameshift insertions
Init Codon
CNVs
a
Loss
c
b d
Phosphorylation
and
displacement
G2 Mi
Kinases
Missense
c.481G>T
Exon 4
Splicing
c.3705-2A>G
Exons 25-26
Missense
c.5654T>C
Exon 35
D D
PCM PCM
Proteinaceus linker
CROCC
M
CROCC Structure
Chr 1: 16,921,950-16,972,979 (1p36.13)
Fw strand
Tot_51kb
M
RC2
RC1
0
400
800
1200
RC1 RC2
Somatic
coding mutations
Nu
mb
er
ofm
uta
tio
ns
Rhabdoid Colorectal Cancers
RC2 RC1
50µm
Rhabdoid Colorectal Cancer
RC1
1056 1078
CROCC 0%
KIF6 3%
DNAH12 2%
DNAH7 5%
ENAH 1%
EPPK1 4%
DMD 15%
NEXN 2%
SPIRE2 1%
SPTBN4 5%
TTN 37%
MCM3AP 5%
MUS81 0.5%
ERCC6 5%
PFAS 0.5%
PML 1%
PSMD3 3%
CDC27 3%
CTDP1 1%
KIAA1543 1%
Copy Gain Missense Mutation
Copy Loss Truncating Mutation
Colorectal Cancer TCGA data set N=224
20µm
20µm
20µm
Figure 1
a
50µm
RC2
CR
OC
C
im
munola
belli
ng
Fallopian Tube
20 µm
Control
Normal mucosa NM1
CR
OC
C
im
munola
belli
ng
pooled exons 5-7 and 33-35
0
100
200
300
400R
ela
tive C
RO
CC
m
RN
A l
evels
**
*
NM1 NM2 RC1 RC2
Mitotic
catastrophe
Centrosome
failure
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a
c
RC5
k
20 µm 40 µm
RC4
j
20 µm Chr 12 Chr 17
RC1
a
10 µm
Cyto
log
ical
an
om
alie
s
d
Figure 2
0 Negative
1 Single
2 Together
2 Separate
00.10.20.30.40.50.6
RC
1R
C2
RC
3R
C4
RC
5R
C6
RC
7
Cells
with >
2 s
ignals
(adju
ste
d r
atio)
20 µm
1.0
0.8
0.6
0.4
0.2
0.0
H&
E
b
CE
N
sig
nal (%
)
0
1
2
>2
1.0
0.8
0.6
0.4
0.2
0.0
RC3 RC4 RC5 RC6 RC7
RC1 RC2 RC3 RC4 RC5 RC6 RC7
CIMP
BRAFV600E
MSI
RAS
TP53
APC
SMARCB1
proximal
median
distal 1p36.1
3
LO
H
Altera
tion
s
Absent
Present
Absent
Present
*
0
100
200
300
400
500
600
3 4 5 6 7 3 4 5 6 7
Rela
tive C
RO
CC
mR
NA
le
ve
ls
Normal Tumor
**
**
**
**
plo
idy
Dapi CEN HER2
CK18 CROCC CROCC
CR
OC
C
imm
unola
belli
ng
(%
)
RC5
KI67
Dapi CEN HER2
Chr 17
Dapi CEN MDM2
CROCC IHC signals
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a
Amplification
Mutation
mRNA Up
mRNA Down
Del
Pediatric Rhabdoid (N=20)
Copy Number
Alterations
Mutation
load
mRNA
Figure 3 c
RI 4 0 Negative
1 Single
2 Together
2 Separate
Rhabdoid of infants (RI)
1 2 3
4
7 5 6
b
e
**
RI
CE
N
sig
nal (%
)
RC
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
0
1
2
>2
RI
800
600
400
200
0
CR
OC
C
imm
unola
belli
ng (
%)
H&
E
50 µm
20µm
20µm
40µm Rela
tive
CR
OC
C
tran
script le
vels
CR
OC
C
DA
PI
CE
N C
hr
1
SM
AR
CB
1
RI
Wnt/β-catenin APC
CTNNB1
MAPK
KRAS
NRAS
HRAS
EGFR
BRAF
ERBB2
ERBB4
PI(3)K
PIK3CA
PTEN
AKT1
Genome
integrity
TP53
ATM
MLH1
Chromatin
modifiers
SMARCB1
EZH2
Others
CDKN2A
CDH1
MET
GNAQ
RET
SMAD4
FBXW7
IDH1
MPL
GNAS
PTPN11
RB1
CSF1R
HNF1A
FGFR1
JAK2
NPM1
SMO
FGFR2
JAK3
NOTCH1
SRC
FGFR3
IDH2
PDGFRA
STK11
FLT3
KDR
ALK
GNA11
KIT
VHL
RI RC
45%
5%
10%
0%
0%
d
MIssesnse
Truncating
Mutations
Pathways
1.0
0.8
0.6
0.4
0.2
0.0
0 30 60 90
RC
RI
P=0.048
log-rank test
Overa
ll surv
ival
Time (months)
median (months)
6 (n=7)
20 (n=7)
CROCC
CROCC IHC signals
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c
d
CON CROCC KD
H&
E
20 µm
10 µm
10 µm
Figure 4
a DAPI α-TUB
DAPI α-TUB γ-TUB DAPI α-TUB CROCC
DAPI α-TUB γ-TUB
CON CROCC KD
5 µm
meta
ph
ase
2N 4N
ACA DAPI
ACA DAPI
CON CROCC KD
10 µm **
b
e
CROCC KD
DAPI γ-TUB
DAPI 5um
DAPI γ-TUB CENTRIN DAPI α-TUB γ-TUB
a b
Time (h)
50
40
30
20
10
0
inte
rphase
Segre
ga
tio
n e
rrors
(%
)
n>
100 p
er
cell
line
Micronuclei Monopolar
spindle
CROCC
KD
***
**
CON CROCC
KD
CON
0
0.1
0.2
0.3
0.4
0.5
0 12 24 36 72
OD
(570 n
m)
CON
CROCC KD
0
0.2
0.4
0.6
0.8
1
0 12 24 36 72
OD
(570 n
m)
serum deprivation serum
*
METAPHASE PROMETAPHASE ANAPHASE
Abnormal spindles (85%) Bipolar
0
20
40
60
80
100
N 2N 3N 4N
Meta
ph
ases (
%)
CON n=15 CROCC KD n=18
**
0
0.04
0.08
0.12
0.16
0.2
Tre
trap
loid
y (
%)
4N/2N ratio
NS
** **
** *
*
**
** ***
** **
NS
*
*
*
DAPI γ-TUB CENTRIN
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0
10
20
30
40
50
PI
CO
UN
T (
%)
T84
a DAPI γ-TUB α-TUB
020406080
100
T84n=43
T84 SDn=68
Mitotic e
rrors
(%
)
Aanaphase aberrations
Micronuclei* DAPI CROCC
ANAPHASE
0
20
40
60
80
100
0 2 4 6 8 10 12
T84GFP+
T84-CROCC/GFP+
T84-C
RO
CC
/GF
P+
CROCC-GFP DAPI CROCC γ-TUB
GF
P+
CE
LLS
(%
)
Time (Days)
** ** ***
***
50 µm
5 µm
b
***
c
Cells
with ≥
3yH
2A
X
foci (%
)
1.0
0.8
0.6
0.4
0.2
0.0 0
0.04
0.08
0.12
0.16
0.2
4N
/2N
(%
)
MSI
BRAF
(V600E)
MSS
CROCC
reduced
dosage
GROSS MITOTIC
ERRORS
CENTROSOME
COHESION
FAILURE
RHABDOID
d
MIT
OT
IC E
RR
OR
S
CRC WITH VULNERABLE
MICROTUBULE FUNCTION
***
Figure 5
DAPI γ-TUB α-TUB
**
T84/GFP+ T84-CROCC/GFP+
6 days 12 days
ANAPHASE
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Published OnlineFirst May 21, 2018.Mol Cancer Res Andrea Remo, Erminia Manfrin, Pietro Parcesepe, et al. Link Rhabdoid Phenotypes and Lethal Colorectal CancersCentrosome Linker-induced Tetraploid Segregation Errors
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